Amygdala regulates emotional behaviors by processing sensory information and by triggering defensive responses in the lower brain structures. Synaptic plasticity in the sensory inputs to amygdala is believed to be a mechanism for auditory fear conditioning which is a simple model of emotional learning. We investigate how synaptic properties of amygdala inputs are related to emotional learning. In previous years we discovered that mice with the forebrain-restricted knockout of a small GTPase Rap1 are impaired in fear learning and have reduced anxiety. Using whole-cell recording, we also found that this mutation selectively altered synaptic transmission in the cortical, but not in the thalamic input to lateral amygdala.[unreadable] [unreadable] During past fiscal year we were trying to identify physiological mechanisms underlying loss of plasticity in the cortico-amygdala synapses of the Rap1 knockout mice. For the first time, we demonstrated that Rap1 acts pre-synaptically by documenting changes in pre-synaptic properties in this input that might account for the loss of plasticity: 1) increased efficacy of synaptic transmission; 2) higher release probability of glutamate and 3) greater number of vesicles released in response to a single action potential. To investigate cellular and molecule mechanisms whereby Rap1 regulates pre-synaptic release we are testing two hypotheses: 1) rap1 alters size of synaptic terminals thereby changing the amount of transmitter been released, 2) rap1 regulates signaling cascade leading to transmitter release. [unreadable] During last fiscal year we were collaborating with NINDS electron microscopy imaging facility to test the first hypothesis. Using lentivirus mediated labeling of axonal terminals of cortical origin in the amygdala we compared terminals morphology between Rap1 knockout and control mice. Preliminary data indicate that Rap1 KO did not affect size and shape of synaptic terminals. [unreadable] To test the second hypothesis we established optical measuring of pre-synaptic release of neurotransmitter in mouse primary cortical neurons in response to electrical stimulation. Currently, we are testing modulation of the release by changes in intracellular levels of cAMP, which is known to activate Rap1. Preliminary imaging data indicate that deletion of Rap1 alters kinetics of presynaptic release modulation by cAMP. Currently we are investigating molecular pathways linking Rap1 to pre-synaptic release machinery.[unreadable] [unreadable] b) Developmental divergence between thalamic in cortical inputs to amygdala during adolescence[unreadable] [unreadable] While comparing synaptic properties of thalamic and cortical inputs to amygdala in adult and adolescent mice we found developmental changes taking place in the thalamic, but not in the cortical pathway during transition between adolescent and adult mice. These changes included loss of synaptic plasticity and increase in basal release of glutamate in this pathway. We hypothesized that these physiological changes should alter functions of thalamic pathway. To test this hypothesis, we tested effects of functional isolation of thalamic pathway on fear learning in adult and adolescent mice by eliminating alternative cortical pathway. We found that in adolescent mice with cortical lesions thalamic pathway can support fear learning at the same level with that in non-lesioned animals, whereas elimination of cortical pathway resulted in impaired fear learning. These findings suggest that adult and adolescence may utilize different brain structures during fear learning. One important implication of this finding is that neuronal basis of same emotional disorder in adults and adolescents may reside in the different areas of the brain.